Tuesday, March 17, 2015

3 17 2015 DNA Isolation using E.Z.N.A Mollusc DNA Kit

Since last week I found that fresh samples produced the High Molecular Weight DNA I wanted. I've been trying troubleshooting the frozen samples. Today we received a kit from Omega Bioscience that is supposedly optimized for Mollusc DNA which can be troublesome. Today I used 4 samples from the September 2014 collection at Oyster Bay to test this kit for frozen tissues.

The samples were 91912014 1H13-16 9/10/11/12.

First the kit assumes that the samples are stored or processed with liquid nitrogen as it asks you to used a mortar and pestle to grind the tissue into powder. Our samples are not preserved this way so I modified the first step to work with our samples.

Before beginning the procedure I also mixed all the reagents necessary for the kit. I added Absolute Ethanol to the DNA wash and pure Isopropyl alcohol to the HBC buffer in the quantities required. These bottles have been labelled accordingly for future reference.

The protocol is as follows:

  1. Added 350 ul ML1 Buffer
  2. Added 25 ul Proteinase K solution
  3. Used pestle in homogenization tube to grind tissue in solution
  4. Vortexed
  5. Incubated at 60 C for 30 minutes
  6. Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
  7. Vortexed
  8. Centrifuged 10,000 g for 2 minutes
  9. Transferred the upper aqueous phase to new tube (~300 ul)
  10. Added 300 ul MBL Buffer
  11. Added 10 ul RNase A
  12. Vortexed for 15 seconds
  13. Incubated at 70C (started at 67.5 C) for 10 minutes
  14. Cooled to room temperature sitting for 5 minutes
  15. Added 300 ul 100% EtOH
  16. Vortexed for 15 seconds
  17. Put spin column in collection tube
  18. Added 750 ul sample solution to column
  19. Centrifuged at 10,000 g for 1 minute
  20. Discarded flowthrough
  21. Repeated 18-20 with remaining sample
  22. Discarded collection tube and replaced with a new one. 
  23. Added 500 ul HBC solution. 
  24. Centrifuged at 10,000 g for 30 seconds
  25. Discarded flowthrough
  26. Added 700 ul DNA Wash Buffer
  27. Centrifuged at 10,000 g for 1 minute
  28. Discarded flowthrough
  29. Repeated 26-28
  30. Centrifuged Empty column for 2 minutes at 10,000 g
  31. Discarded collection tube and put column into microcentrifuge tube for sample collection
  32. Added 100 ul preheated 70C Elution Buffer
  33. Incubated for 2 minutes
  34. Centrifuged at 10,000 g for 1 minute
  35. Repeated 32-34. 
  36. Stored DNA at -20 C
Tomorrow I will run the DNA out on a gel to check for quality. 

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